NCT00001174 In order to better interpret the impact of genetic variation on the brain biology of bipolar disorder (BD), we are pursuing a variety of functional genomics studies, including brain imaging, RNA-sequencing in post-mortem brain tissue, and cellular phenotyping of neurons derived from induced pluripotent stem cells. We have contributed genotypes and imaging data to the Enhancing NeuroImaging Genetics through Meta-Analysis (ENIGMA) brain imaging consortium, which brings together imaging and genetics data from around the world to generate large sample sizes needed for robust results. In addition, enrollees in our ongoing Amish Mennonite Bipolar Genetics study (AMBiGen, ZIA-MH002843) are referred for diffusion tensor imaging thru the Amish Connectome Study. Neuroimaging endophenotypes may shed light on the mechanisms whereby common genetic variants influence risk for psychiatric disorders. We have completed RNA-sequencing in 200 postmortem brain samples obtained from people with and without major psychiatric disorders (BD, schizophrenia and major depression). This study focused on the subgenual anterior cingulate cortex, especially implicated in people with mood disorders. The results illustrate the enormous diversity of brain-expressed transcripts, demonstrate that major psychiatric disorders can be distinguished at the transcript level, and suggest that alternative splicing is one mechanism through which differences in gene expression arise in the brain. Ongoing studies in this sample include experimental validation of differentially expressed genes and transcripts, analysis of co-expression networks, and single-cell RNA sequencing, which will help identify cell-type specific genes and transcripts that are dysregulated in mental illness. We also seek to model the functional genomics of disease-related genes in cells derived from induced pluripotent stem cell (iPSC) lines. This project aims to explore the ways in which we can use iPSC technology to study the biological impact of genes and genetic mutations that we identify in our other ongoing studies. Working with the NIH Center for Regenerative Medicine, the National Institute of Neurological Disorders and Stroke (NINDS) and the National Heart, Lung and Blood Institute (NHLBI) stem cell cores we have so far successfully reprogrammed fibroblasts into iPSCs from 72 AMBiGen participants. We are developing a large iPSC-based resource and associated work-flows that constitute a living catalog of psychiatric risk alleles. iPSC-derived cells are studied with high-resolution microscopic imaging, electrophysiology, and gene expression methods. These data could reveal differences between control and patient-derived cells and the impact of known and novel therapeutic agents. In collaboration with Joseph Steiner (NINDS), we are developing a morphologic assay to measure the effect of known therapeutics and stressors on dendritic number and length following differentiation of NPCs into neurons. We are also exploring ways to measure the functional impact of genetic mutations at the cellular level and to use genome editing tools such as CRISPR-Cas9 to rescue cellular phenotypes and establish a causal role for specific genetic mutations. In a recently published study (Jiang et al. 2019) we used neural cells from carriers of GWAS-identified common risk alleles on chromosome 3p22 to test the influence of these alleles on gene expression and transcription factor binding. We also explored the impact of widely-used mood stabilizing medications. The results showed that neural cells carrying risk alleles displayed lower baseline expression of a nearby gene, TRANK1, that was rescued by chronic treatment with therapeutic dosages of valproic acid (VPA). In collaboration with NIH Distinguished Scientist Gary Felsenfeld, we demonstrated that a nearby SNP strongly affects binding by the transcription factor, CTCF. Decreased expression of TRANK1 perturbed expression of many genes involved in neural development and differentiation. This proof-of-concept study demonstrates the value of iPSC-based assays for translating even common, low-risk alleles identified by GWAS into novel genetic, neurobiological, and pharmacological insights. Through SNP array screening of cases enrolled in AMBiGen, we have identified a family in which 3 individuals carry a rare duplication event on chromosome 16p11.2 known to confer risk for BD and other illnesses. iPSC lines have been established for all 3 carriers and 3 non-carrier relatives and have been differentiated into neural cells. Transcriptomic analyses indicate that some genes in the duplicated region show increased expression in neurons, but that many other genes are also dysregulated in CNV carriers. Overexpressed genes are enriched for several pathways, including neuronal growth and proliferation, MAP kinase signaling, and cell migration. Ongoing experiments are aimed at testing the hypothesis that MAP kinase inhibitors can ameliorate the morphological changes. If successful, these experiments could form the basis for future collaborations aimed at screening for novel therapeutics. In the coming year, we will collect iPSC lines from additional families. We also plan to apply single-cell sequencing technology to iPSC-derived cells to explore variation in DNA sequence and gene expression across individual cells from the same donor, which can be an important source of variation in human iPSC studies. The above approaches may illuminate factors that contribute to disruption of biological mechanisms during the disease process and provide cell-based assays for high throughput screening for novel therapeutics. Multigenic disorders such as BD pose special challenges for experimental studies, since a single causative mutation is usually not identifiable. Thus we are also studying rare, single-gene disorders whose symptoms overlap with those seen in common mental illnesses. Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by behavioral abnormalities and disruptions in circadian rhythm. Cells from people living with SMS obtained in collaboration with Ann Smith (NHGRI) have been reprogrammed into iPSCs and differentiated into neurons and other brain cells. We are using these cells to explore the biochemical and molecular characteristics of SMS. Early results suggest that SMS mutations have a major impact on the growth, morphology, and transcriptome of developing neurons. Findings from this study may have relevance to other neuropsychiatric disorders with circadian rhythm disturbances, such as depression and BD. In the coming year, we will continue studies in neural cells derived from people with BD, SMS, and unaffected relatives. If successful, these projects will help unpack the biology behind GWAS results, identify high-risk alleles, and shed new light on how risk alleles act within neural cells to generate biological changes in the brain. The findings may identify new targets that lead to better methods of diagnosis and treatment for mood and anxiety disorders.